Antibacterial activities and mechanisms of action of a defensin from manila clam Ruditapes philippinarum.

Defensins represent an evolutionary ancient family of antimicrobial peptides, which played an undeniably important role in host defense. In the present study, a defensin isoform was identified and characterized from manila clam Ruditapes philippinarum (designed as Rpdef1α). Multiple alignments and phylogenetic analysis suggested that Rpdef1α belonged to the defensin family. Quantitative RT-PCR and immunohistochemical analysis revealed that Rpdef1α transcripts and the encoding peptide were dominantly expressed in the tissues of gills and mantle. After Vibrio anguillarum challenge, the Rpdef1α transcripts were significantly up-regulated in gills of clams. In addition, rRpdef1α not only showed broad-spectrum antimicrobial activities towards Vibrio species, but also inhibited the formation of bacterial biofilms. Knockdown of Rpdef1α transcripts caused significant increase in the cumulative mortality of manila clams post V. anguillarum challenge. Membrane integrity, scanning electron microscopy analysis and electrochemical assay indicated that rRpdef1α was capable of causing bacterial membrane permeabilization and then resulted in cell death. Moreover, phagocytosis and chemotactic ability of hemocytes could be significantly enhanced after incubation with rRpdef1α. Overall, these results suggested that Rpdef1α could act as both antibacterial agent and opsonin to defend against the invading microorganisms in manila clam R. philippinarum.